巴西苏木素通过抑制SarA以PIA依赖性方式减少MRSA生物膜形成的研究  

Study on the reduction of MRSA biofilm formation by Brazilin through the inhibition of SarA in a PIA-dependent manner

作　　者：李明哲 杨智芳 周小仙 刘和兰 黎瑞 周永雯 陈泽慧 Li Mingzhe;Yang Zhifang;Zhou Xiaoxian;Liu Helan;Li Rui;Zhou Yongwen;Chen Zehui(Department of Laboratory Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563003;School of Laboratory Medicine,Zunyi Medical University,Zunyi 563006;The Second Affiliated Hospital of Zunyi Medical University,Zunyi 563000)

机构地区：[1]遵义医科大学附属医院医学检验科,遵义563003 [2]遵义医科大学检验医学院,遵义563006 [3]遵义医科大学第二附属医院,遵义563000

出　　处：《中国抗生素杂志》2024年第5期527-534,共8页Chinese Journal of Antibiotics

基　　金：贵州省科学技术厅科学技术基金项目(No.黔科合支撑【2021】一般034);遵义市科技计划项目(No.黔科平台人才【2020】-025);遵义市科技计划项目(No.遵市科合HZ字【2021】299号)。

摘　　要：目的 探究巴西苏木素(brazilin,BN)抑制耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)生物膜形成的机制,为开展中医药防治MRSA感染提供新思路。方法 建立MRSA ATCC43300菌株生物膜模型,试验分为BN 8、16和32μg/mL组及对照组;通过结晶紫法测定BN对MRSA生物膜的抑制作用;刚果红平板法、硫酸-苯酚法检测BN对细胞间多糖黏附素(polysaccharide intercellular adhesion,PIA)合成的影响;使用real-time PCR(qRT-PCR)方法验证胞间黏附素操纵子icaA和icaD转录水平;分析高通量转录组测序可能存在的生物膜抑制基因;通过qRT-PCR、Western blot方法对相关基因及蛋白加以验证;通过皮下埋植静脉导管建立MRSA生物膜感染小鼠模型进一步验证BN疗效。结果 与对照组相比,不同浓度的BN组均能显著减少MRSA生物膜及PIA生成,其中在最低剂量8μg/mL BN作用下,MRSA生物膜形成量下降40.17%(P<0.01)、PIA减少55.56%(P<0.01);通过qRT-PCR验证不同浓度的BN组均能显著抑制icaA和icaD基因的表达,其中在8μg/mL浓度下,icaA和icaD基因的表达量分别比对照组降低了59.03%(P<0.01)、48.33%(P<0.05);高通量测序显示sarA下调-1.458 log2;qRTPCR、Western blot结果显示,BN能显著抑制sarA转录及其蛋白表达;通过体内实验显示,BN能有效抑制小鼠体内MRSA生物膜的形成。结论 BN通过抑制SarA的表达,减少PIA的合成,进而干预生物膜的形成。Objective The goal was to find out how Brazilin(BN) inhibited methicillin-resistant Staphylococcus aureus(MRSA) from making biofilms and to give new ideas for using traditional Chinese medicine to prevent and treat MRSA infections.Methods A biofilm model of the MRSA ATCC43300 strain was established.The experiment was divided into BN 8,16 and 32 μg/mL groups as well as a control group.The inhibitory effect of BN on MRSA biofilm was determined through the crystal violet method.The Congo red agar plate method and the sulfuric acid-phenol method were employed to detect the effect of BN on the synthesis of polysaccharide intercellular adhesion(PIA).Quantitative real-time PCR(qRT-PCR) was used to verify the transcription levels of the icaA and icaD gene clusters involved in intercellular adhesion.High-throughput transcriptomic sequencing was performed to identify potential biofilm-inhibitory genes.qRT-PCR and Western blot validated the relevant genes and proteins.The mouse model of MRSA biofilm infection through subcutaneous placement of an intrvenous catheter was established to further validate the efficacy of BN.Results Compared to the control group,BN at different concentrations significantly reduced MRSA biofilm and PIA formation.Specifically,at the lowest dose of 8 μg/mL BN,MRSA biofilm formation was decreased by 40.17%(P<0.01) and PIA by 55.56%(P<0.01).qRT-PCR verification indicated that BN at different concentrations significantly inhibited the expression of the icaA and icaD genes.Compared to the control group,the expression levels of icaA and icaD genes were reduced by 59.03%(P<0.01) and 48.33% at an 8 μg/mL concentration(P<0.05),respectively.High-throughput sequencing revealed a sarA downregulation of-1.458 log2.qRT-PCR and Western blot results showed that BN significantly inhibited the transcription and protein expression of sarA.In vivo experiments indicated that BN effectively inhibited MRSA biofilm formation in mice.Conclusion BN reduces the synthesis of PIA by inhibiting the expression of SarA and the

关 键 词：巴西苏木素 耐甲氧西林金黄色葡萄球菌 生物膜 PIA SARA 

分 类 号：R978[医药卫生—药品]